Abstract
Purpose :
Very-long-chain polyunsaturated fatty acids (VLC-PUFAs) are a class of non-dietary lipids found in the retina and a few other tissues in vertebrates. VLC-PUFAs comprise <2% of the fatty acids in the retina and are depleted in retinal disorders such as diabetic retinopathy, age-related macular degeneration, and Stargardt 3 disease (STGD3). Their exact roles in the retina and contribution to retinal disease pathology are largely unknown. Certain mutations in exon 6 of ELOVL4 that cause STGD3 lead to premature termination and truncation of the ELOVL4 protein, which causes a loss of catalytic activity and cytoplasmic aggregation. As mouse models of global ELOVL4-ablation are not useful due to neonatal skin drying, we turned to zebrafish to understand the effects of VLC-PUFA depletion and protein mislocalization on vision. We hypothesized that mutations in zebrafish Elovl4 would lead to the loss of ocular VLC-PUFAs, lipid abnormalities, and visual dysfunction.
Methods :
Using CRISPR-Cas9, we induced deletions without neonatal mortality in exon 2 of Elovl4b (knockout of VLC-PUFA synthesis), exon 3 of Elovl4a (knockout of very-long-chain saturated fatty acid synthesis), and a homology-driven repair insertion of the two-1-bp-deletion mutation in exon 7 of Elovl4b (STGD3 knock-in). This complex mutation was first identified in a Utah family with STGD3. We confirmed germline mutagenesis by sequencing the gene loci in F2 progeny. We performed visual motor response (VMR) tests using a ViewPoint ZebraBox system.
Results :
Homozygous Elovl4b knockout (KO) fish had depleted ocular levels of C30-C36 VLC-PUFAs and significant depletions in glycerophospholipids containing C30 and C32 VLC-PUFAs, but an upregulation of C26 acylcarnitine. They had no difference in gross retinal morphology but had abnormal lipid deposits in their retinal pigment epithelium. Homozygous Elovl4a knockout fish were infertile and smaller than heterozygous mutants and wild-type controls. Homozygous STGD3 knock-in fish are viable and fertile but are exclusively female. All Elovl4 mutants displayed varying VMR phenotypes between 5-6 days post-fertilization.
Conclusions :
We generated viable zebrafish models of STGD3 and Elovl4-ablation. The models can be used to understand STGD3 pathology and differentiate the role of VLC-PUFA depletion from STGD3 protein aggregation in visual function and photoreceptor viability.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.